Persistent infection of mink with Aleutian mink disease parvovirus (ADV) leads to progressive immune disorder characterized by high levels of antiviral antibodies, hypergammaglobulinemia, plasmacytosis, and immune complex disease. Virus is not neutralized in vivo and ADV exists in infectious immune complexes. Isolates of ADV differ markedly in their ability to induce AD; some like ADV-Utah and ADV-TR are highly pathogenic in vivo, but are replication defective for CRFK cells. The cell-culture ADV-G is replication competent for CRFK, but replicates poorly, if at all, in mink and does not cause progressive disease. A major goal of this project is to correlate specific DNA sequences of the ADV genome to functional correlates, such as pathogenicity determinants and strain variation. Another major goal is to study the actual structure of the ADV virion. Understanding of the structure at high resolution will enable us to (a) map epitopes and pathogenicity determinants to discreet coordinates on the viral particle, (b) relate structural features to the unique biology of ADV in vivo. A summary of the results obtained within the last year includes: ? Structural studies of ADV. Further refinement of the ADV VP2-only capsid structure using cryo-EM has revealed that the novel protrusions found abutting the 3-fold axes are comprised of short sequences from non-adjacent regions of 2 protomers of VP2. These knobs overlay the most antigenic surface regions of parvoviruses and may partly account for the unusual immunological properties of ADV. Cryo-EM studies of the VP1:VP2::10:1 particle revealed that this particle differs from the VP2 only capsid in several features, primarily a smoothing of the of the 3-fold protrusions. Monoclonal antibodies that aggregate particles (mAB#165) or bind without aggregating (mAB#Y-2-9) have been identified. Crystals of VP2-only capsids have been obtained that diffract to a resolution of 8 Angstroms. ? Identification of putative cellular receptor for ADV. Using a virion overlay protein binding assay (VOPBA), we have identified a 67 kD protein (ABP binding protein, ABP) on the membrane of CRFK cells that binds 125I-labeled VP2 capsids of ADV with high affinity. Virus does not bind to Sf-9 or Mus dunni cells in the VOPBA. The binding is specific and can be competed out by unlabeled ADV capsids, but not by capsids from an unrelated non-enveloped virus (CCMV), and is sensitive to neuraminidase. Partially purified ABP blocks ADV binding to CRFK cells. ABP is a likely candidate for the cellular receptor for ADV. ? Mapping determinants of ADV host range and pathogenicity. We continued to study full-length molecular clones chimeric between ADV-G and the pathogenic ADV-Utah. Three separate point mutations in ADV VP2 from the ADV-G to the ADV-Utah residue (G83D, H395Q, N434H) each abrogate replication in CRFK cells. Two additional mutations to the ADV-Utah residues (I352V, H534D) conferred the ability to replicate in vivo and induce high levels of antibody, but these constructs did not induce typical AD. An additional change to the ADV-Utah residue (N491E) produced a virus that was replication competent in vitro, but not infectious in vivo.